Method for purifying marine collagen and the processing thereof into porous sponges

ABSTRACT

Methods are provided for purifying marine collagen and for processing the collagen into porous sponges. Products produced with these methods and the use of the products are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 11/817,044 which is a Section 371 of International Application No.PCT/EP2006/001313, filed Feb. 14, 2006, which was published in theGerman language on Aug. 31, 2006, under International Publication No. WO2006/089660 A2, and the disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates to a method for purifying marine collagen,especially for enhancing the smell and appearance of marine collagen.

The invention further relates to a method for the production of poroussponges of marine collagen which are suitable in particular for medicalpurposes.

Collagen is a biodegradable as well as biocompatible protein which isused as a starting material for manifold applications in the foodindustry, in the pharmaceutical and cosmetic industries as well as inmedicine.

“Marine collagen” is understood to mean collagen isolated from marineorganisms, that is, organisms living in the sea, especially from membersof the Porifera strain, preferably from Chondrosia reniformis.

A large number of collagen products are known and being utilized inmedicine. Such products include, for example, sponges, fibers ormembranes.

The vast majority of these products is manufactured from collagen whichis produced from the connective tissue, the skin, the bones, or thetendons of mammals, for example from cattle, horses or pigs. Theessential disadvantages of these products are to be seen in the factsthat:

in most cases, a young animal has to be used as the source for thecollagen in order to obtain a sufficient yield of collagen;

the collagen obtained is in most cases soluble both in acid media aswell as in basic media, which necessitates an additional crosslinkingreaction (physically, by heat treatment, or chemically, by usingbifunctional substances) in order to enhance the mechanical propertiesof the collagen and its stability in liquid media;

there is a risk of contamination with bovine spongiform encephalopathy(BSE).

As an alternative to producing collagen from mammals, Internationalpatent application publication WO 01/64046 describes a method forisolating collagen from marine sponges of the genus Chondrosiareniformis (Porifera, Demospongiae).

In this method, fresh sponge starting material is imbibed in alcohol andthen washed with water, and an extracting agent is added thereto,preferably at a pH of 7-12. The resultant collagen extract is processedby increasing the pH of the suspension to a value of 8-11, stirring,centrifuging, subsequently lowering the pH value of the supernatant, aswell as centrifuging and isolating the precipitate.

The sponge collagen obtainable by this method does, however, havedisadvantages consisting essentially in that:

the collagen solution has a dirty appearance;

the collagen is microbiologically impure; and

products from this sponge collagen have an unpleasant smell.

BRIEF SUMMARY OF THE INVENTION

The task underlying the present invention thus was to eliminate theabove-mentioned disadvantages of marine collagen and to provide collagenfrom marine organisms, which is also suitable for the manufacture ofproducts for medicinal purposes.

This task is solved by a method wherein a collagen precipitate ispurified by using chemical treatment steps, thereby enhancing the smelland appearance thereof. The purified collagen can subsequently beprocessed into porous sponges by lyophilization.

According to the present invention, the method for purifying marinecollagen encompasses treating a collagen precipitate, as obtainable, forexample, according to the method described in WO 01/64046, with hydrogenperoxide in aqueous solutions at different pH values.

To this end, the moisture of the collagen precipitate should initiallybe adjusted to a content of 70 to 95% by weight, preferably to a contentof 80 to 90% by weight. This can be accomplished by wringing thecollagen precipitate under pressure or by centrifugation, in order toreduce the moisture content of the collagen precipitate to the desiredcontent, if necessary.

The collagen precipitate is then suspended in an aqueous H₂O₂ solutioncontaining 0.1 to 1% (v/v), preferably 0.5% (v/v), of H₂O₂. Then the pHof the collagen suspension is adjusted to a value of from 11 to 13. Thiscauses the collagen to dissolve.

Following incubation of the collagen with H₂O₂ under alkalineconditions, the collagen solution is filtered to remove insolublecomponents. Thereafter, the collagen is precipitated from the solutionby adding a suitable water-miscible organic solvent to the solution.

The collagen precipitate is filtered off, and its moisture content isreadjusted to 70-95% by weight, preferably 80 to 90% by weight. Then,the collagen is again dissolved in an aqueous H₂O₂ solution, which has acontent of H₂O₂ of 0.1 to 1% (v/v), preferably of 0.5% (v/v), and the pHof this collagen solution is adjusted to a value of from 5 to 7, so thatthe collagen may then be used for the production of sponges, membranesor fibers for medical purposes.

Adjustment of the pH to a value of from 11 to 13 is preferablyaccomplished with NaOH, but it may also be accomplished with otheralkali hydroxides or alkaline earth hydroxides, such as KOH, Ca(OH)₂ orMg(OH)₂.

As water-miscible organic solvents, ethanol and isopropanol arepreferred. Precipitation is preferably performed at a ratio of water toethanol which is between 1:2 and 1:3.

Neutralization of the collagen solution to a pH of 5 to 7 may beaccomplished by reducing the pH value by adding organic acids, e.g.,formic acid, acetic acid, citric acid, ascorbic acid, propionic acid,lactic acid, or inorganic acids, such as hydrochloric acid, phosphoricacid or sulfuric acid. Preferably, the pH value is adjusted by additionof 5 N hydrochloric acid.

The advantages of the method according to the invention are:

a high yield in protein extraction;

a sterile collagen which is free of bacteria;

a BSE-free collagen since Porifera have no neural structures;

a collagen with a pleasing appearance which does not look dirty;

a collagen without unpleasant smell;

a collagen which is insoluble in acid media and which does notnecessitate an additional crosslinking reaction in order to improve itsmechanical properties and stability in liquids; and

a collagen which can be used for the production of dressings for woundhealing or as a support in tissue regeneration.

On account of its advantages, the collagen purified by using the methodaccording to the present invention is particularly suitable for themanufacture of products for medical purposes.

Therefore, the collagen purified using the method according to thepresent invention, as well as the use thereof for the manufacture ofporous sponges, fiber material or membranes, are also subject matters ofthe present invention.

The present invention thus also relates to methods for the production ofporous sponges from the purified marine collagen.

Porous collagen sponges may be produced by a method wherein a solutionobtained according to the method of the present invention is deep-frozenand freeze-dried after the collagen solution has either been foamed upby vigorous shaking/stirring or after the air contained in the collagensolution has been removed in vacuo, in order to produce sponges withsmaller pores. The collagen solution preferably contains collagen at aconcentration of 0.5 to 5% by weight.

By using appropriate molds, in which the collagen solution or thecollagen foam is frozen, it is possible to produce porous collagen foamsof any shape.

Collagen sponges with further enhanced properties, especially with aview to their medical application, can be obtained by acid treatment ofthe collagen sponges after these have been freeze-dried. Acid treatmentis performed with inorganic acids, preferably by immersing the sponge inan 0.1 NHCl solution. Thereafter, the acid-treated sponge can bedehydrated by deep-freezing and freeze-drying, or by immersing inethanol and air-drying.

Employing the method according to the present invention for producingcollagen sponges, it is possible also to produce porous collagen spongeswith antimicrobial properties. To this end, an antimicrobially activesubstance can be added to the collagen suspension prior todeep-freezing. A preferred antimicrobially active substance is silversulfadiazine, which is added in an amount of 1% by weight, relative tothe amount of collagen in the suspension.

As an alternative to the afore-described approach, the porous collagenfoams can be immersed, after the acid treatment, in a solutioncontaining an antimicrobially active substance. Subsequently, thecollagen sponge can be dehydrated by freeze-drying or, if theantimicrobially active substance is insoluble in ethanol, by immersingin ethanol and subsequent air-drying.

Additional subject matters of the present invention are therefore theporous collagen sponges produced by the aforementioned method and theuse of the sponges for the manufacture of products for medicinalpurposes, such as wound dressings.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in greater detail with reference tothe following specific, non-limiting examples.

Example 1 Method for Purifying Sponge Collagen

A collagen precipitate precipitated in an acid medium (pH 3), which hadbeen obtained in accordance with the method described in WO 01/64046,was separated from the medium by filtration and wrung out under pressureuntil a residual moisture content of approximately 84% by weight wasobtained. The collagen fibers obtained in this manner were freeze-driedfor storage at a temperature of approx. −20° C.

121 g of the frozen collagen fibers were suspended in 1300 ml of aqueous0.5% (v/v) H₂O₂ solution while stirring for 2 hours. Then, the pH of thesolution was adjusted to a value of 12.4 with a 5N solution of NaOH, inorder to dissolve the collagen fibers. The resulting collagen solutionwas filtered to remove insoluble components and then poured into 2600 mlethanol (conc. 98%), while stirring vigorously. The collagen therebyprecipitated was of a white or slightly yellowish color and had afibrous appearance.

The collagen fibers were freed from the medium by filtration, wrung outunder pressure or by centrifugation and subsequently homogenouslysuspended in 300 ml of an aqueous 0.5% (v/v) H₂O₂ solution, whilestirring. The pH of the solution was adjusted to a value of 6.5 with a 5N HCL solution. Adjustment of the pH was performed while stirringvigorously, in order to avoid formation of fiber precipitates. In thismanner, a sterile collagen solution with a collagen concentration of2.8% by weight was obtained.

All method steps were performed at room temperature, and all objectscoming into contact with the collagen were rinsed with an 0.5% by weightH₂O₂ solution before being used.

Example 2 Production of Porous Sponges Using Purified Sponge Collagen

A solution of sponge collagen with a collagen concentration of 2% byweight and a pH value of 6.5, which had been obtained using a methodaccording to Example 1, was foamed by vigorous stirring with the aid ofan Ultra Turrax. The collagen foam was cast into a rectangular mold madeof polypropylene or polystyrene. The heights of the foam layers variedbetween 2 and 8 mm. The collagen foam was frozen in the mold at −40° C.and then lyophilized.

Example 3 Production of Sponges on the Basis of Precipitated SpongeCollagen

Porous sponges of sponge collagen were produced as described in Example2. Subsequently, the freeze-dried sponges were subjected to a 30 minuteacid treatment by immersion in an 0.1 N HCl solution. Following thistreatment, the sponges were repeatedly washed with distilled water untilthe last residues of acid were removed.

These sponges were frozen in a still moist state and dried bylyophilization.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A marine collagen purified in accordance with a method comprising thesteps: adjusting a moisture content of a collagen precipitate, which hasbeen precipitated in an acid medium, to 70 to 95% by weight; suspendingthe precipitated collagen in a first aqueous H₂O₂ solution; solubilizingthe suspended collagen by adjusting a pH of the collagen suspension to avalue of from 11 to 13; reprecipitating the suspended collagen with anorganic, water-miscible solvent; filtering off of the reprecipitatedcollagen; adjusting a moisture content of the reprecipitated collagen to70 to 95% by weight; resuspending the reprecipitated collagen in asecond aqueous H₂O₂ solution; and adjusting the pH of the resuspendedcollagen to a value of from 5 to
 7. 2. A porous sponge produced from themarine collagen of claim 1, wherein the porous sponge is produced inaccordance with a production method comprising the steps: foaming asolution of the marine collagen or degassing the solution of the marinecollagen in vacuo; optionally placing the solution of the marinecollagen in a mold; deep-freezing the foamed or degassed collagensolution, thereby forming a collagen sponge; and lyophilizing thecollagen sponge.
 3. The porous sponge according to claim 2, wherein thesolution has a collagen content of 0.5 to 5% by weight.
 4. The poroussponge according to claim 2, further comprising an antimicrobiallyactive substance in the sponge.
 5. The porous sponge according to claim4, wherein the antimicrobially active substance is present in an amountof about 1% by weight, relative to the collagen content of the sponge.6. The porous sponge according to claim 4, wherein the antimicrobiallyactive substance comprises silver sulfadiazine.
 7. The porous spongeaccording to claim 2, wherein the production method further comprisestreating the collagen sponge, subsequent to the lyophilization thereof,with an inorganic acid, and thereafter washing out the collagen spongewith water until there is no longer any acid detectable.
 8. The poroussponge according to claim 7, wherein the inorganic acid is anapproximately 0.1 N HCl solution.
 9. The porous sponge according toclaim 2, having a form of a medical product.